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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 17 August 2010 and 14 September 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Sponsor's identification: Novares LA 300 (phenol, methylstyrenated)
Description : pale amber coloured slightly viscous liquid
Batch number : 28166
Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
Date received : 16 April 2010
Expiry date : 31 December 2010
Storage conditions: room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test Species
A mixed population of sewage sludge micro-organisms was obtained on 17 August 2010 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2-free air* for approximately 1 hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.4 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 litres) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.

* CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules
Duration of test (contact time):
28 d
Initial conc.:
23.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
METHODS
The test item was poorly water soluble and so, following the recommendations of the International Standards Organisation (ISO 1995) and at the request of the Sponsor, the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper and addition to the test vessels. Using this method enables relatively small amounts of test item to be accurately added to each test vessel.
An amount of test item (1014 mg) was dissolved in tetrahydrofuran (10 ml) to give a 1014 mg/10 ml solvent stock solution. For each test vessel, an aliquot (25 µl) of this solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate to dryness for approximately 30 minutes. Inoculum (350 ml) was dispersed in culture medium to a final volume of 3.5 litres and aliquots (107 ml) of the inoculated culture medium were dispensed to each of 29 replicate vessels (125 ml glass Wheaton bottles) prior to the addition of the test item coated filter paper to give a test concentration of 23.7 mg/l, equivalent to 20 mg carbon/l. The vessels were sealed using Teflon lined silicon septa and aluminium crimp caps.
A test concentration of 20 mg carbon/l was employed in the study following the recommendations of the test guidelines.
Control vessels (33 replicates) were prepared each containing inoculated culture medium and a filter paper. Tetrahydrofuran (25 µl) was dispensed onto each filter paper and evaporated to dryness for approximately 30 minutes in order to maintain consistency between the control and the test item vessels.

* Whatman GF/A (21 mm diameter)

Data from the control vessels was shared with similar concurrent studies.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration, or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa) (Sigma Lot No. 096K1231), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the reference item directly in culture medium. An aliquot (137.2 ml) of this stock solution was dispersed with inoculum (400 ml) and culture medium, final volume 4 litres, to give a test concentration of 34.3 mg/l, equivalent to 20 mg carbon/l. Aliquots (107 ml) of the 34.3 mg/l test concentration were dispensed to each of 33 replicate test vessels (125 ml glass Wheaton bottles). A filter paper* onto which tetrahydrofuran
(25 µl) had been dispensed and evaporated to dryness for approximately 30 minutes was added to each vessel in order to maintain consistency between the test and reference item vessels. The vessels were sealed using Teflon lined silicon septa and aluminium crimp caps.
The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.


Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the study.
An aliquot (34.3 ml) of the reference item stock solution was dispersed with inoculum (100 ml) and culture medium, final volume 1 litre, to give a reference item concentration of 34.3 mg/l, equivalent to 20 mg carbon/l. Aliquots (107 ml) of the 34.3 mg/l reference item concentration were dispensed to 9 replicate test vessels. An aliquot (25 µl) of the test item solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate to dryness for approximately 30 minutes prior to addition to the inoculated culture medium to give the required test item concentration of 23.7 mg/l, equivalent to 20 mg carbon/l. Each test vessel was sealed using Teflon lined silicon septa and aluminium crimp caps.
The final concentration in the toxicity control vessels was 23.7 mg test item/l plus 34.3 mg reference item/l, equivalent to 40 mg carbon/l.

* Whatman GF/A (21 mm diameter).

Culture medium
The culture medium used in this study (see Appendix 1 in any other information on materials and method section) was that recommended in the OECD and ISO Guideline.

Preparation of test system
The following test preparations were prepared and incubated in 125 ml glass Wheaton bottles (total volume when full 160 ml) each containing 107 ml of solution:
a) A control consisting of inoculated culture medium, plus a filter paper*, 33 replicate vessels.
b) The reference item (sodium benzoate) in inoculated culture medium, plus a filter paper*, to give a final concentration of 20 mg carbon/l, 33 replicate vessels.
c) The test item, on a filter paper*, in inoculated culture medium, to give a final concentration of 20 mg carbon/l, 29 replicate vessels.
d) The test item, on a filter paper*, plus the reference item in inoculated culture medium, to give a final concentration of 40 mg carbon/l to act as a toxicity control, 9 replicate vessels.
Test media a-d were inoculated with the prepared inoculum at a final concentration of 100 ml/l.
Aliquots (107 ml) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the control, reference item, test item, and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional control and reference item vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated in darkness at 20 ± 1oC with constant shaking at approximately 125 rpm (INFORS Version 2 Multitron® Incubator).

* Whatman GF/A (21 mm diameter) with 25 µl of tetrahydrofuran evaporated off.

Sampling and analysis

Inorganic carbon analysis
CO2 production in the vessels was determined by measuring the increase in the concentration of Inorganic Carbon (IC) in the headspace.
Triplicate control, reference item and test item vessels were sacrificed on days 0, 2, 6, 8, 10, 14, 16 and 21 for IC analysis. On day 28, five replicate vessels were sacrificed for IC analysis. Triplicate toxicity control vessels were sacrificed on days 0, 8 and 14 for IC analysis.
An aliquot (1.0 ml) of concentrated orthophosphoric acid was injected through the septum of each vessel taken for analysis in order to lower the pH of the medium to <3*. The vessels were then shaken at approximately 150 rpm (INFORS TR-225 orbital platform shaker and INFORS Version 2 Multitron® Incubator) for 1 hour at 20±1oC prior to samples being withdrawn from the headspace and analysed for IC.
The principle of this method is that after acidification to a pH value of <3 and equilibration at 20 ± 1oC, the equilibrium constant for the distribution of CO2 between the liquid and gaseous phases in the test vessels is 1.0 and hence only the CO2 concentration in the headspace needs to be determined.
The samples were analysed using a Shimadzu TOC-VCSH TOC analyser. Samples (50 µl) taken from the headspace were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

*Confirmed by preliminary work conducted where the pH of 107 ml of culture medium containing varying concentrations of Na2CO3, acidified by the addition of 1.0 ml concentrated orthophosphoric acid, was determined to be between 0.6 and 0.8.

Dissolved organic carbon (DOC) analysis
On Days 0 and 28 samples (20 ml) were removed from the control and reference item vessels prepared for DOC analysis and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test item and toxicity control vessels was not possible due to the insoluble nature of the test item in water.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 Deg C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

Evaluation of data
For calculation of carbon content and percentage degradation see in attached section.

Validation criteria
Test items giving a result of ≥ 60% yield of ThIC within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation exceeding 10%.
The test is considered valid if the reference item degradation rate is ≥ 60% by day 14.
The toxicity control should attain ≥ 25% degradation by day 14 for the test item to be considered as non-inhibitory.
The TIC produced from the control bottles at the end of the test should be ≤ 15% of the TOC added initially as test item.

Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable.
Test performance:
The mean TIC in the control vessels on Day 28 was 0.86 mg/l; equivalent to 4% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.
The test item attained 4% degradation after 28 days and hence cannot be considered to be readily biodegradable.
The toxicity control attained 38% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.
Parameter:
% degradation (CO2 evolution)
Value:
4
Sampling time:
28 d
Details on results:
The test item was poorly water soluble and so, following the recommendations of the International Standards Organisation (ISO 1995) and at the request of the Sponsor, the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper and addition to the test vessels.
Total inorganic carbon values for the test item, reference item, toxicity control, and control vessels at each analysis occasion are given in Table 1 (see in Any other information on results incl. tables section). Percentage biodegradation values for the test and reference items and the toxicity control are given in Table 2 (see in Any other infromation on results incl. tables section) and the biodegradation curves are presented in Figure 1 (see in attached section). The results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 3 (see in Any other information on results incl. tables section).
Results with reference substance:
Sodium benzoate attained 83% degradation after 14 days and 89% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 (see Table 3) showed that the replicate reference item vessels attained 96% and 94% degradation for each replicate vessel. The degradation rates for the reference item were slightly higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO2 evolution occurring.

Table 1              Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg TIC)

Reference Item (mg TIC)

Test Item (mg TIC)

Toxicity Control

(mg TIC)

R1

R2

R3

R4

R5

R1

R2

R3

R4

R5

R1

R2

R3

R4

R5

R1

R2

R3

0

0.07

0.05

0.05

-

-

0.05

0.05

0.05

-

-

0.09

0.05

0.05

-

-

0.07

0.07

0.07

2

0.11

0.07

0.08

-

-

1.33

1.47

1.34

-

-

0.07

0.07

0.07

-

-

-

-

-

6

0.07

0.06

0.07

-

-

1.35

1.34

1.40

-

-

0.06

0.06

0.05

-

-

-

-

-

8

0.07

0.06

0.07

-

-

1.63

1.59

1.54

-

-

0.08

0.07

0.08

-

-

1.45

1.51

1.31

10

0.09

0.07

0.08

-

-

1.78

1.92

1.87

-

-

0.08

0.07

0.07

-

-

-

-

-

14

0.09

0.07

0.09

-

-

1.86

1.99

1.71

-

-

0.06

0.07

0.09

-

-

1.81

1.61

1.71

16

0.11

0.07

0.08

-

-

1.74

1.88

1.95

-

-

0.05

0.07

0.06

-

-

-

-

-

21

0.10

0.09

0.08

-

-

1.79

2.11

1.73

-

-

0.06

0.07

0.07

-

-

-

-

-

28

0.11

0.08

0.09

0.09

0.09

2.01

2.00

1.97

2.13

2.05

0.34

0.10

0.08

0.08

0.07

-

-

-

R


R1– R5= Replicates 1 to 5

- = No value determined

Table 2              Percentage Biodegradation Values

Day

% Degradation

Reference Item

Test Item

Toxicity Control

0

0

0

0

2

60

0

-

6

60

0

-

8

71

0

32

10

83

0

-

14

83

0

38

16

83

0

-

21

84

0

-

28

89

4

-

 


  - = No value determined

Table 3              Dissolved Organic Carbon (DOC) Values in the Control and Reference Item Vessels on Days 0 and 28

DOC Concentration

Test Vessel

Day 0

Day 28

mg C/l

mg C/l Corrected for Mean Control Value

% of Nominal Carbon Content

mg C/l

mg C/l Corrected for Mean Control Value

% Degradation

Control

R1

2.58

-

-

1.31

-

-

 

R2

1.31

-

-

1.21

-

-

Reference Item

R1

20.97

19.02

95

2.06

0.80

96

 

R2

20.84

18.89

95

2.40

1.14

94


R1– R2= Replicates 1 and 2

 

 REFERENCE

International Standards Organisation (ISO), ISO 10634 (1995) Water Quality – Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item attained 4% degradation after 28 days and therefore cannot be considered to be readily biodegradable.
Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No. 310 and the ISO Guideline No 14593 “Water quality – Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2headspace test)”.

Methods.

The test item was poorly water soluble and so, following the recommendations of the International Standards Organisation (ISO 1995) and at the request of the Sponsor, the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper and addition to the test vessels. This test was conducted in sealed vessels and assessed the biodegradation of the test item by measuring the inorganic carbon present in the headspace of the vessels. 

The test item, at a concentration of 20 mg C/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 20 ± 1°C for 28 days.

The degradation of the test item was assessed by the determination of carbon dioxide produced on Days 0, 2, 6, 8, 10, 14, 16, 21 and 28. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test item attained 4% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Conclusion.

The test item attained 4% degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given; comparable to guideline study; test was performed by an laboratory with an high reputation for delivery of solid data.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2,4-diphenyl-4-methyl-1-pentene, 4-methyl-2,4-diphenylpent-1-ene
- no further information on test material
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sludge was sampled and prepared according to Japanes standard procedure (samples taken from 10 different sites: 3 city sewage plants, 1 industrial sewage plant, 3 rivers and 3 bays; sampling 4 times every year)
- Laboratory culture: sludge was kept in culture for 3 months
- Method of cultivation: Sludge was cultured at 25 ± 2°C. On every day about 30 min after cessation of aeration, 1/3 of the supernatant was removed and replaced by an equal amount of dechlorinated water. Aeration was continued and synthetic sewage (5% w/v of glucose, peptone and monopotassium phosphate, pH adjusted to 7 ± 1 with sodium hydroxide) was added.
- Storage conditions: at 25 ± 2°C
- Storage length: 3 month
- Preparation of inoculum for exposure: measurement of concentration of suspended solid according to JIS K 0102 method 14.1
Duration of test (contact time):
4 wk
Initial conc.:
100 other: ppm
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS (see CITI 1992)
- Composition of medium: 3 mL of solution A, B, and C as described in JIS K 0102 method 21 were made up to 1000 mL with purified water and the pH was adjusted to 7.0
- Test temperature: 25 ± 2
- pH: 7
- pH adjusted: if required
- Aeration of dilution water: no data
- Suspended solids concentration: 100 ppm
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Close system oxygen consumption measuring apparatus; 300 mL glas vessels
- Number of culture flasks/concentration: 3 for test substance, 2 for test substance blanks, 1 for abiotic control, 1 for positive control
- Method used to create aerobic conditions: production of O2 as O2 was consumed.
- Measuring equipment: Coulometer Ohkura Electric Co., Ltd.
- Test performed in closed system: yes
- Details of trap for CO2 and volatile organics if used: soda lime No. 1 (extra pure reagent, Wako Pure Chemical Industries, Ltd.)
- Other: test solution was stirred by a magnetic stirrer

SAMPLING
- Sampling frequency: continuous registration of O2 consumption; test substance determination at the beginning and at the end of the test

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic control: yes
- Toxicity control: no
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
4 wk
Parameter:
% degradation (test mat. analysis)
Remarks:
HPLC
Value:
3
Sampling time:
4 wk
Validity criteria fulfilled:
yes
Remarks:
; criteria as required by MITI-I method
Interpretation of results:
under test conditions no biodegradation observed
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given; comparable to guideline study; test was performed by an laboratory with an high reputation for delivery of solid data.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4-(1-methyl-1-phenylethyl)phenol [synonym:2-phenyl-2-(4-hydroxyphenyl)propane]
- no further information on test material
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sludge was sampled and prepared according to Japanese standard procedure (samples taken from 10 different sites: 3 city sewage plants, 1 industrial sewage plant, 3 rivers and 3 bays; sampling 4 times every year)
- Laboratory culture: sludge was kept in culture for 3 month
- Method of cultivation: Sludge was cultured at 25 ± 2°C. On every day about 30 min after cessation of aeration, 1/3 of the supernatant was removed and replaced by an equal amount of dechlorinated water. Aeration was continued and synthetic sewage (5% w/v of glucose, peptone and monopotassium phosphate, pH adjusted to 7 ± 1 with sodium hydroxide) was added.
- Storage conditions: at 25 ± 2°C
- Storage length: 3 month
- Preparation of inoculum for exposure: measurement of concentration of suspended solid according to JIS K 0102 method 14.1
Duration of test (contact time):
4 wk
Initial conc.:
100 other: ppm
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS (see CITI 1992)
- Composition of medium: 3 mL of solution A, B, and C as described in JIS K 0102 method 21 were made up to 1000 mL with purified water and the pH was adjusted to 7.0
- Test temperature: 25 ± 2
- pH: 7
- pH adjusted: if required
- Aeration of dilution water: no data
- Suspended solids concentration: 100 ppm
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Close system oxygen consumption measuring apparatus; 300 mL glas vessels
- Number of culture flasks/concentration: 3 for test substance, 2 for test substance blanks, 1 for abiotic control, 1 for positive control
- Method used to create aerobic conditions: production of O2 as O2 was consumed.
- Measuring equipment: Coulometer Ohkura Electric Co., Ltd.
- Test performed in closed system: yes
- Details of trap for CO2 and volatile organics if used: soda lime No. 1 (extra pure reagent, Wako Pure Chemical Industries, Ltd.)
- Other: test solution was stirred by a magnetic stirrer

SAMPLING
- Sampling frequency: continuous registration of O2 consumption; test substance determination at the beginning and at the end of the test

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic control: yes
- Toxicity control: no
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
4 wk
Parameter:
% degradation (test mat. analysis)
Remarks:
HPLC
Value:
7
Sampling time:
4 wk
Validity criteria fulfilled:
yes
Remarks:
; criteria as required by MITI-I method
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
In a MITI-I test (OECD 301C) for ready biodegradability performed under the Japanese Chemical Inspection & Testing Institute, no biodegradation of the test substance 4-(α,α-dimethylbenzyl)phenol (p-cumylphenol) was observed.

Description of key information

In valid screening studies for ready biodegradability, no significant biodegradation was found, either with the entire UVCB substance Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol [EC no. 700-960-7] or with two main constituents, the dimeric non-phenolic constituent, 4-methyl-2,4-diphenylpent-1-ene [CAS no. 6362-80-7] and the phenolic constituent p-cumylphenol [CAS no. 599-64-4].

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information